Nucleotide sequences which code for the zwa1 gene

ABSTRACT

The invention relates to an isolated polynucleotide comprising a polynucleotide sequence chosen from the group consisting of  
     a) polynucleotide which is identical to the extent of at least 70% to a polynucleotide which codes for a polypeptide which comprises the amino acid sequence of SEQ ID NO 2,  
     b) polynucleotide which codes for a polypeptide which comprises an amino acid sequence which is identical to the extent of at least 70% to the amino acid sequence of SEQ ID NO 2  
     c) polynucleotide which is complementary to the polynucleotides of a) and b) and  
     d) polynucleotide comprising at least 15 successive nucleotides of the polynucleotide sequence of a), b) or c),  
     and processes for the fermentative preparation of L-amino acid with amplification of the zwa1 gene in the coryneform bacteria employed.

INTRODUCTION AND BACKGROUND

[0001] The invention provides nucleotide sequences, which code for thezwa1 gene and processes for the fermentative preparation of amino acids,in particular L-lysine, using coryneform bacteria in which the zwa1 geneis amplified. All references cited herein are expressly incorporated byreference. Incorporation by reference is also designated by the term“I.B.R.” following any citation.

PRIOR ART

[0002] Amino acids, in particular L-lysine, are used in human medicineand in the pharmaceuticals industry, but in particular in animalnutrition. It is known that amino acids are prepared by fermentationfrom strains of coryneform bacteria, in particular Corynebacteriumglutamicum. Because of their great importance, work is constantly beingundertaken to improve the preparation processes. Improvements to theprocesses can relate to fermentation measures, such as e.g. stirring andsupply of oxygen, or the composition of the nutrient media, such as e.g.the sugar concentration during the fermentation, or the working up tothe product form by e.g. ion exchange chromatography, or the intrinsicoutput properties of the microorganism itself.

[0003] Methods of mutagenesis, selection and mutant selection are usedto improve the output properties of these microorganisms. Strains whichare resistant to antimetabolites, such as e.g. the lysine analogueS-(2-aminoethyl)-cysteine, or are auxotrophic for metabolites ofregulatory importance and produce L-amino acids are obtained in thismanner.

[0004] Recombinant DNA techniques have also been employed for some yearsfor improving the strain of Corynebacterium strains which produce aminoacids, by amplifying individual amino acid biosynthesis genes andinvestigating the effect on the amino acid production.

[0005] Review articles in this context are to be found, inter alia, inKinoshita (“Glutamic Acid Bacteria”, in: Biology of IndustrialMicroorganisms, Demain and Solomon (Eds.) I.B.R., Benjamin Cummings,London, UK, 1985, 115-142), Hilliger (BioTec 2, 40-44 (1991)) I.B.R.,Eggeling (Amino Acids 6:261-272 (1994)) I.B.R., Jetten and Sinskey(Critical Reviews in Biotechnology 15, 73-103 (1995)) I.B.R. and Sahm etal. (Annuals of the New York Academy of Science 782, 25-39 (1996))I.B.R.

OBJECT OF THE INVENTION

[0006] An object of the invention is to provide new measures forimproved fermentative preparation of amino acids, in particularL-lysine. Amino acids, in particular L-lysine, are used in humanmedicine, in the pharmaceuticals industry and in particular in animalnutrition. There is therefore a general interest in providing newimproved processes for the preparation of amino acids, in particularL-lysine. When L-lysine or lysine are mentioned in the following, notonly the base but also the salts, such as e. g. lysine monohydrochlorideor lysine sulfate, are also meant by this.

SUMMARY OF THE INVENTION

[0007] The new DNA sequence of C. glutamicum which codes for the zwa1gene and which as a constituent of the present invention is SEQ ID NO 1and related sequences. The amino acid sequence of the corresponding geneproduct of the zwa1 gene has furthermore been derived from the presentDNA sequence. The resulting amino acid sequence of the zwa1 gene productis SEQ ID NO 2 and related sequences.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008]FIG. 1: Map of the plasmid pCR2.1zwa1exp The length data are to beunderstood as approximate values.

DETAILED DESCRIPTION OF THE INVENTION

[0009] The invention provides an isolated polynucleotide from coryneformbacteria, comprising a polynucleotide sequence chosen from the groupconsisting of

[0010] a) polynucleotide which is identical to the extent of at least70% to a polynucleotide which codes for a polypeptide which comprisesthe amino acid sequence of SEQ ID NO 2,

[0011] b) polynucleotide which codes for a polypeptide which comprisesan amino acid sequence which is identical to the extent of at least 70%to the amino acid sequence of SEQ ID NO 2,

[0012] c) polynucleotide which is complementary to the polynucleotidesof a) or b), and

[0013] d) polynucleotide comprising at least 15 successive nucleotidesof the polynucleotide sequence of a), b) or c).

[0014] The invention also provides a polynucleotide according to claim1, this preferably being a DNA which is capable of replication,comprising:

[0015] (i) the nucleotide sequence shown in SEQ ID NO 1, or

[0016] (ii) at least one sequence which corresponds to sequences (i)within the range of the degeneration of the genetic code, or

[0017] (iii) at least one sequence which hybridizes with sequencescomplementary to sequences (i) or (ii), and optionally

[0018] (iv) sense mutations of neutral function in (i).

[0019] The relative degree of substitution or mutation in thepolynucleotide or amino acid sequence to produce a desired percentage ofsequence identity can be established or determined by well-known methodsof sequence analysis. These methods are disclosed and demonstrated inBishop, et al. “DNA & Protein Sequence Analysis (A Practical Approach”),Oxford Univ. Press, Inc. (1997) I.B.R. and by Steinberg, Michael“Protein Structure Prediction” (A Practical Approach), Oxford Univ.Press, Inc. (1997) I.B.R. Hybridization of complementary sequences canoccur at varying degrees of stringency. Sambrook et al.: MolecularCloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989)I.B.R.

[0020] The invention also provides

[0021] a polynucleotide, comprising the nucleotide sequences as shown inSEQ ID NO 1, wherein the polynucleotide is a preferably recombinant DNAwhich is capable of replication in coryneform bacteria,

[0022] a polynucleotide, which codes for a polypeptide which comprisesthe amino acid sequence as shown in SEQ ID NO 2,

[0023] a vector containing the DNA sequence of C. glutamicum which codesfor the zwa1 gene, contained in the vector (plasmid) pCR2.1zwa1exp.,deposited in E. coli Top10F′ under number DSM 13115

[0024] and coryneform bacteria serving as the host cell, which containthe vector in which the zwa1 gene is amplified.

[0025] The invention also provides polynucleotides which substantiallycomprise a polynucleotide sequence, which are obtainable by screening bymeans of hybridization of a corresponding gene library, which comprisesthe complete gene with the polynucleotide sequence corresponding to SEQID NO 1 or parts thereof, with a probe which comprises the sequence ofthe said polynucleotide according to SEQ ID NO 1 or a fragment thereof,and isolation of the DNA sequence mentioned.

[0026] Polynucleotide sequences according to the invention are suitableas hybridization probes for RNA, cDNA and DNA, in order to isolate, inthe full length, cDNA which code for the Zwa1 gene product and in orderto isolate those cDNA or genes which have a high similarity of sequencewith that of the zwa1 gene.

[0027] Polynucleotide sequences according to the invention arefurthermore suitable as primers with the aid of which DNA of genes whichcode for the zwa1 gene can be prepared by the polymerase chain reaction(PCR). Such oligonucleotides which serve as probes or primers compriseat least 30, preferably at least 20, very particularly preferably atlease 15 successive nucleotides. Oligonucleotides which have a length ofat least 40 or 50 nucleotides are also suitable.

[0028] “Isolated” means separated out of its natural environment.

[0029] “Polynucleotide” in general relates to polyribonucleotides andpolydeoxyribonucleotides, it being possible for these to be non-modifiedRNA or DNA or modified RNA or DNA.

[0030] “Polypeptides” is understood as meaning peptides or proteinswhich comprise two or more amino acids bonded via peptide bonds.

[0031] The polypeptides according to the invention include polypeptidesaccording to SEQ ID NO 2, in particular those with the biologicalactivity of the gene product of the zwa1 gene and also those which areidentical to the extent of at least 70% to the polypeptide according toSEQ ID NO 2, preferably to the extent of at least 80%, and in particularwhich are identical to the extent of at least 90% to 95% to thepolypeptide according to SEQ ID NO 2 and have the activity mentioned.

[0032] The invention moreover provides a process for the fermentativepreparation of amino acids, in particular L-lysine, using coryneformbacteria which in particular already produce the amino acid, and inwhich the nucleotide sequences which code for the zwa1 gene areamplified, in particular over-expressed.

[0033] The term “amplification” in this connection describes theincrease in the intracellular activity of one or more enzymes in amicroorganism which are coded by the corresponding DNA, for example byincreasing the number of copies of the gene or genes, using a potentpromoter or using a gene which codes for a corresponding enzyme having ahigh activity, and optionally combining these measures.

[0034] The microorganisms, which the present invention provides, canprepare L-lysine from glucose, sucrose, lactose, fructose, maltose,molasses, starch, cellulose or from glycerol and ethanol. They can berepresentatives of coryneform bacteria, in particular of the genusCorynebacterium. Of the genus Corynebacterium, there may be mentioned inparticular the species Corynebacterium glutamicum, which is known amongexperts for its ability to produce L-amino acids.

[0035] Suitable strains of the genus Corynebacterium, in particular ofthe species Corynebacterium glutamicum, are, for example, the knownwild-type strains

[0036]Corynebacterium glutamicum ATCC13032

[0037]Corynebacterium acetoglutamicum ATCC15806

[0038]Corynebacterium acetoacidophilum ATCC13870

[0039]Corynebacterium melassecoloa ATCC17965

[0040]Corynebacterium thermoaminogenes FERM BP-1539

[0041]Brevibacterium flavum ATCC14067

[0042]Brevibacterium lactofermentum ATCC13869 and

[0043]Brevibacterium divaricatum ATCC14020

[0044] and L-lysine-producing mutants or strains prepared therefrom,such as, for example

[0045]Corynebacterium glutamicum FERM-P 1709

[0046]Brevibacterium flavum FERM-P 1708

[0047]Brevibacterium lactofermentum FERM-P 1712

[0048]Corynebacterium glutamicum FERM-P 6463

[0049]Corynebacterium glutamicum FERM-P 6464 and

[0050]Corynebacterium glutamicum DSM5715

[0051] The inventors have succeeded in isolating the new zwa1 gene of C.glutamicum which codes for the Zwa1 gene product. To isolate the zwa1gene or also other genes of C. glutamicum, a gene library of thismicroorganism is first set up in E. coli. The setting up of genelibraries is described in generally known textbooks and handbooks. Thetextbook by Winnacker: Gene und Klone, Eine Einführung in dieGentechnologie [Genes and Clones, An Introduction to GeneticEngineering] (Verlag Chemie, Weinheim, Germany, 1990) I.B.R. or thehandbook by Sambrook et al.: Molecular Cloning, A Laboratory Manual(Cold Spring Harbor Laboratory Press, 1989) I.B.R. may be mentioned asan example. A well-known gene library is that of the E. coli K-12 strainW3110 set up in λ vectors by Kohara et al. (Cell 50, 495-508 (1987))I.B.R. Bathe et al. (Molecular and General Genetics, 252:255-265, 1996)I.B.R. describe a gene library of C. glutamicum ATCC13032, which was setup with the aid of the cosmid vector SuperCos I (Wahl et al., 1987,Proceedings of the National Academy of Sciences USA, 84:2160-2164)I.B.R. in the E. coli K-12 strain NM554 (Raleigh et al., 1988, NucleicAcids Research 16:1563-1575) I.B.R.

[0052] Börmann et al. (Molecular Microbiology 6(3), 317-326 (1992))I.B.R. in turn describe a gene library of C. glutamicum ATCC13032 usingthe cosmid pHC79 (Hohn and Collins, Gene 11, 291-298 (1980)) I.B.R. Toprepare a gene library of C. glutamicum in E. coli it is also possibleto use plasmids such as pBR322 (Bolivar, Life Sciences, 25, 807-818(1979)) I.B.R. or pUC9 (Vieira et al., 1982, Gene, 19:259-268) I.B.R.Suitable hosts are, in particular, those E. coli strains, which arerestriction- and recombination-defective.

[0053] An example of these is the strain DH5αMCR, which has beendescribed by Grant et al. (Proceedings of the National Academy ofSciences USA, 87 (1990) 4645-4649) I.B.R. The long DNA fragments clonedwith the aid of cosmids can then in turn be subcloned and subsequentlysequenced in the usual vectors which are suitable for sequencing, suchas is described e. g. by Sanger et al. (Proceedings of the NationalAcademy of Sciences of the United States of America, 74:5463-5467, 1977)I.B.R.

[0054] The new DNA sequence of C. glutamicum which codes for the zwa1gene and which is a constituent of the present invention as SEQ ID NO 1was obtained in this manner. The amino acid sequence of thecorresponding gene product of the zwa1 gene has furthermore been derivedfrom the present DNA sequence. The resulting amino acid sequence of thezwa1 gene product is shown in SEQ ID NO 2.

[0055] Coding DNA sequences, which result from SEQ ID NO 1 by thedegeneracy of the genetic code, are also a constituent of the invention.In the same way, DNA sequences which hybridize with SEQ ID NO 1 or partsof SEQ ID NO 1 are a constituent of the invention. Conservative aminoacid exchanges, such as e. g. an exchange of glycine for alanine or ofaspartic acid for glutamic acid in proteins, are furthermore known amongexperts as “sense mutations” which do not lead to a fundamental changein the activity of the protein, i.e. are of neutral function.

[0056] It is furthermore known that changes on the N and/or C terminusof a protein cannot substantially impair or can even stabilize thefunction thereof. Information in this context can be found by theexpert, inter alia, in Ben-Bassat et al. (Journal of Bacteriology169:751-757 (1987)) I.B.R., in O'Regan et al. (Gene 77:237-251 (1989))I.B.R., in Sahin-Toth et al. (Protein Sciences 3:240-247 (1994)) I.B.R.,in Hochuli et al. (Bio/Technology 6:1321-1325 (1988)) I.B.R. and inknown textbooks of genetics and molecular biology. Amino acid sequenceswhich result in a corresponding manner from SEQ ID NO 2 are also aconstituent of the invention.

[0057] In the same way, DNA sequences which hybridize with SEQ ID NO 1or parts of SEQ ID NO 1 are a constituent of the invention. Finally, DNAsequences which are prepared by the polymerase chain reaction (PCR)using primers which result from SEQ ID NO 1 are a constituent of theinvention. Such oligonucleotides typically have a length of at least 15nucleotides.

[0058] Instructions for identifying DNA sequences by means ofhybridization can be found by the expert, inter alia, in the handbook“The DIG System Users Guide for Filter Hybridization” from BoehringerMannheim GmbH (Mannheim, Germany, 1993) I.B.R. and in Liebl et al.(International Journal of Systematic Bacteriology (1991) 41: 255-260)I.B.R. Instructions for amplification of DNA sequences with the aid ofthe polymerase chain reaction (PCR) can be found by the expert, interalia, in the handbook by Gait: Oligonukleotide synthesis: a practicalapproach (IRL Press, Oxford, UK, 1984) I.B.R. and in Newton and Graham:PCR (Spektrum Akademischer Verlag, Heidelberg, Germany, 1994) I.B.R.

[0059] The inventors have found that coryneform bacteria produce aminoacids, in particular L-lysine, in an improved manner afterover-expression of the zwa1 gene. To achieve an over-expression, thenumber of copies of the corresponding genes can be increased, or thepromoter and regulation region or the ribosome binding site upstream ofthe structural gene can be mutated. Expression cassettes which areincorporated upstream of the structural gene act in the same way. Byinducible promoters, it is additionally possible to increase theexpression in the course of fermentative amino acid production.

[0060] The expression is likewise improved by measures to prolong thelife of the m-RNA. Furthermore, preventing the degradation of the enzymeprotein also increases the enzyme activity. The genes or geneconstructions can either be present in plasmids with a varying number ofcopies, or can be integrated and amplified in the chromosome.Alternatively, an over-expression of the genes in question canfurthermore be achieved by changing the composition of the media and theculture procedure.

[0061] Instructions in this context can be found by the expert, interalia, in Martin et al. (Bio/Technology 5, 137-146 (1987)) I.B.R., inGuerrero et al. (Gene 138, 35-41 (1994)) I.B.R., Tsuchiya and Morinaga(Bio/Technology 6, 428-430 (1988)) I.B.R., in Eikmanns et al. (Gene 102,93-98 (1991)) I.B.R., in European Patent Specification EPS 0 472 869I.B.R., in U.S. Pat. No. 4,601,893 I.B.R., in Schwarzer and Pühler(Bio/Technology 9, 84-87 (1991) I.B.R., in Reinscheid et al. (Appliedand Environmental Microbiology 60, 126-132 (1994)) I.B.R., in LaBarre etal. (Journal of Bacteriology 175, 1001-1007 (1993)) I.B.R., in PatentApplication WO 96/15246 I.B.R., in Malumbres et al. (Gene 134, 15-24(1993)) I.B.R., in Japanese Laid-Open Specification JP-A-10-229891I.B.R., in Jensen and Hammer (Biotechnology and Bioengineering 58,191-195 (1998)) I.B.R., in Makrides (Microbiological Reviews 60:512-538(1996)) I.B.R. and in known textbooks of genetics and molecular biology.

[0062] By way of example, the zwa1 gene according to the invention wasduplicated or over-expressed with the aid of the integration method suchas is described e. g. in Reinscheid et al. (Applied and EnvironmentalMicrobiology 60, 126-132 (1994)) I.B.R. In this method, the completegene is cloned in a plasmid vector, which can replicate in a host(typically E. coli) but not in C. glutamicum. Possible vectors are, forexample, pSUP301 (Simon et al., Bio/Technology 1, 784-791 (1983))I.B.R., pK18mob oder pK19mob (Schäfer et al., Gene 145, 69-73 (1994))I.B.R., pGEM-T (Promega corporation, Madison, Wis., USA) I.B.R.,pCR2.1-TOPO (Shuman (1994) I.B.R. Journal of Biological Chemistry269:32678-84; U.S. Pat. No. 5,487,993) I.B.R., pCR®Blunt (Invitrogen,Groningen, Holland; Bernard et al., Journal of Molecular Biology, 234:534-541 (1993)) I.B.R. or pEM1 (Schrumpf et al, 1991, Journal ofBacteriology 173:4510-4516) I.B.R. The plasmid vector, which containsthe gene to be amplified, is then transferred into the desired strain ofC. glutamicum by conjugation or transformation. The method ofconjugation is described, for example, by Schäfer et al. (Applied andEnvironmental Microbiology 60, 756-759 (1994)) I.B.R.

[0063] Methods for transformation are described, for example, byThierbach et al. (Applied Microbiology and Biotechnology 29, 356-362(1988)) I.B.R., Dunican and Shivnan (Bio/Technology 7, 1067-1070 (1989))I.B.R. and Tauch et al. (FEMS Microbiological Letters 123, 343-347(1994)) I.B.R. After homologous recombination by means of a “cross over”event, the resulting strain contains two copies of the gene in question.The strain DSM5715:: pCR2.1zwa1exp, which carries two copies of the zwa1gene, was prepared in this manner with the aid of the integrationplasmid pCR2.1zwa1exp.

[0064] In addition, it may be advantageous for the production of aminoacids, in particular L-lysine, to amplify, in particular toover-express, one or more enzymes of the particular biosynthesis pathwayof glycolysis, of anaplerosis, of the citric acid cycle or of amino acidexport, in addition to the Zwa1 gene product.

[0065] It may thus be advantageous, for example for the preparation ofL-lysine, for one or more of the genes chosen from the group consistingof

[0066] the dapA gene which codes for dihydrodipicolinate synthase (EP-B0 197 335) I.B.R.,

[0067] the lysC gene which codes for a feed back resistant aspartatekinase

[0068] the dapD gene which codes for tetradihydrodipicolinatesuccinylase (Wehrmann et al., Journal of Bacteriology 180, 3159-3165(1998)) I.B.R.,

[0069] the dapE gene which codes for succinyl diaminopimelatedesuccinylase (Wehrmann et al., Journal of Bacteriology 177: 5991-5993(1995)) I.B.R.,

[0070] the gap gene which codes for glyceraldehyde 3-phosphatedehydrogenase (Eikmanns (1992). Journal of Bacteriology 174:6076-6086)I.B.R.,

[0071] the pyc gene which codes for pyruvate carboxylase (Eikmanns(1992). Journal of Bacteriology 174:6076-6086) I.B.R.,

[0072] the mqo gene which codes for malate:quinone oxidoreductase(Molenaar et al., European Journal of Biochemistry 254, 395-403 (1998))I.B.R., the lysE gene which codes for lysine export (DE-A-195 48 222)I.B.R.

[0073] to be over-expressed at the same time.

[0074] For the production of amino acids, in particular L-lysine, it mayfurthermore be advantageous to attenuate, in addition to the zwa1 gene,

[0075] the gene which codes for phosphate pyruvate carboxykinase (DE 19950 409.1; DSM 13047) I.B.R. and/or

[0076] the pgi gene which codes for glucose 6-phosphate isomerase (U.S.Ser. No. 09/396,478; DSM 12969) I.B.R.

[0077] at the same time.

[0078] In addition to over-expression of the zwa1 gene it mayfurthermore be advantageous, for the production of amino acids, inparticular L-lysine, to eliminate undesirable side reactions, (Nakayama:“Breeding of Amino Acid Producing Microorganisms”, in: Overproduction ofMicrobial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press,London, UK, 1982) I.B.R.

[0079] In addition to amplification of the zwa1 gene, it may beadvantageous to attenuate the zwa2 gene or the action of the associatedgene product of the zwa2 gene. The corresponding gene and the associatedmeasures are to be found in Patent Application 199 59 327.2 I.B.R. filedin parallel.

[0080] An integration vector suitable for insertion mutagenesis,pCR2.1zwa2int, has been deposited under no. DSM13113 in E. coli TOP10F′.

[0081] Plasmid pCR2.1zwa2int comprises the plasmid pCR2.1-TOPO describedby Mead et al. (Bio/Technology 9:657-663 (1991)) I.B.R., into which aninternal fragment of the zwa2 gene, shown in SEQ-ID-No. 1 of GermanPatent Application 199 59 327.2 I.B.R., has been incorporated. Aftertransformation and homologous recombination in the chromosomal zwa2 gene(insertion), this plasmid leads to a total loss of function. By way ofexample, the strain DSM5715::pCR2.1zwa2int, the Zwa2 gene product ofwhich is eliminated, was prepared in this manner.

[0082] The microorganisms prepared according to the invention can becultured continuously or discontinuously in the batch process (batchculture) or in the fed batch (feed process) or repeated fed batchprocess (repetitive feed process) for the purpose of production of aminoacids, in particular L-lysine. A summary of known culture methods isdescribed in the textbook by Chmiel (Bioprozesstechnik 1. Einführung indie Bioverfahrenstechnik [Bioprocess Technology 1. Introduction toBioprocess Technology (Gustav Fischer Verlag, Stuttgart, 1991)) I.B.R.or in the textbook by Storhas (Bioreaktoren und periphere Einrichtungen[Bioreactors and Peripheral Equipment] (Vieweg Verlag,Braunschweig/Wiesbaden, 1994)) I.B.R.

[0083] The culture medium to be used must meet the requirements of theparticular strains in a suitable manner. Descriptions of culture mediafor various microorganisms are contained in the handbook “Manual ofMethods for General Bacteriology” of the American Society forBacteriology (Washington D.C., USA, 1981) I.B.R.

[0084] Sugars and carbohydrates, such as e. g. glucose, sucrose,lactose, fructose, maltose, molasses, starch and cellulose, oils andfats, such as e. g. soya oil, sunflower oil, groundnut oil and coconutfat, fatty acids, such as e. g. palmitic acid, stearic acid and linoleicacid, alcohols, such as e. g. glycerol and ethanol, and organic acids,such as e. g. acetic acid, can be used as the source of carbon. Thesesubstances can be used individually or as a mixture.

[0085] Organic nitrogen-containing compounds, such as peptones, yeastextract, meat extract, malt extract, corn steep liquor, soya bean flourand urea, or inorganic compounds, such as ammonium sulphate, ammoniumchloride, ammonium phosphate, ammonium carbonate and ammonium nitrate,can be used as the source of nitrogen. The sources of nitrogen can beused individually or as a mixture.

[0086] Phosphoric acid, potassium dihydrogen phosphate or dipotassiumhydrogen phosphate or the corresponding sodium-containing salts can beused as the source of phosphorus. The culture medium must furthermorecomprise salts of metals, such as e. g. magnesium sulfate or ironsulfate, which are necessary for growth.

[0087] Finally, essential growth substances, such as amino acids andvitamins, can be employed in addition to the abovementioned substances.Suitable precursors can moreover be added to the culture medium. Thestarting substances mentioned can be added to the culture in the form ofa single batch, or can be fed in during the culture in a suitablemanner.

[0088] Basic compounds, such as sodium hydroxide, potassium hydroxide,ammonia or aqueous ammonia, or acid compounds, such as phosphoric acidor sulfuric acid, can be employed in a suitable manner to control thepH. Antifoams, such as e. g. fatty acid polyglycol esters, can beemployed to control the development of foam. Suitable substances havinga selective action, such as e. g. antibiotics, can be added to themedium to maintain the stability of plasmids.

[0089] To maintain aerobic conditions, oxygen or oxygen-containing gasmixtures, such as e. g. air, are introduced into the culture. Thetemperature of the culture is usually 20° C. to 45° C., and preferably25° C. to 40° C. Culturing is continued until a maximum of lysine hasformed. This target is usually reached within 10 hours to 160 hours.

[0090] The analysis of L-lysine can be carried out by anion exchangechromatography with subsequent ninhydrin derivatization, as described bySpackman et al. (Analytical Chemistry, 30, (1958), 1190) I.B.R.beschrieben.

[0091] The following microorganism has been deposited at the DeutscheSammlung für Mikrorganismen und Zellkulturen (DSMZ=German Collection ofMicroorganisms and Cell Cultures, Braunschweig, Germany) in accordancewith the Budapest Treaty: Escherichia coli strain Top10F′/pCR2.1zwa1expas DSM 13115.

[0092] The process according to the invention is used for thefermentative preparation of amino acids, in particular L-lysine. Inaddition to amplification of the zwa1 gene, it may be advantageous toattenuate the zwa2 gene. The corresponding gene or the vector suitablefor the insertion mutagenesis, pCR2.1zwa2int, is deposited in E.coliTOP10F′ under number DSM13113.

EXAMPLES

[0093] The present invention is explained in more detail in thefollowing with the aid of embodiment examples.

Example 1

[0094] Preparation of a Genomic Cosmid Gene Library from Corynebacteriumglutamicum ATCC 13032

[0095] Chromosomal DNA from Corynebacterium glutamicum ATCC 13032 wasisolated as described by Tauch et al., (1995, Plasmid 33:168-179) I.B.R.and partly cleaved with the restriction enzyme Sau3AI (AmershamPharmacia, Freiburg, Germany, Product Description Sau3AI, Code no.27-0913-02). The DNA fragments were dephosphorylated with shrimpalkaline phosphatase (Roche Molecular Biochemicals, Mannheim, Germany,Product Description SAP, Code no. 1758250).

[0096] The DNA of the cosmid vector SuperCos1 (Wahl et al. (1987)Proceedings of the National Academy of Sciences USA 84:2160-2164)I.B.R., obtained from the company Stratagene (La Jolla, USA, ProductDescription SuperCos1 Cosmid Vektor Kit, Code no. 251301) was cleavedwith the restriction enzyme XbaI (Amersham Pharmacia, Freiburg, Germany,Product Description XbaI, Code no. 27-0948-02) and likewisedephosphorylated with shrimp alkaline phosphatase.

[0097] The cosmid DNA was then cleaved with the restriction enzyme BamHI(Amersham Pharmacia, Freiburg, Germany, Product Description BamHI, Codeno. 27-0868-04). The cosmid DNA treated in this manner was mixed withthe treated ATCC13032 DNA and the batch was treated with T4 DNA ligase(Amersham Pharmacia, Freiburg, Germany, Product DescriptionT4-DNA-Ligase, Code no.27-0870-04).

[0098] The ligation mixture was then packed in phages with the aid ofGigapack II XL Packing Extracts (Stratagene, La Jolla, USA, ProductDescription Gigapack II XL Packing Extract, Code no. 200217).

[0099] For infection of the E. coli strain NM554 (Raleigh et al. 1988,Nucleic Acid Research 16:1563-1575) I.B.R. the cells were taken up in 10mM MgSO₄ and mixed with an aliquot of the phage suspension. Theinfection and titering of the cosmid library were carried out asdescribed by Sambrook et al. (1989, Molecular Cloning: A laboratoryManual, Cold Spring Harbor) I.B.R., the cells being plated out on LBagar (Lennox, 1955, Virology, 1:190) I.B.R. with 100 μg/ml ampicillin.After incubation overnight at 37° C., recombinant individual clones wereselected.

Example 2

[0100] Isolation and Sequencing of the zwa1 Gene

[0101] The cosmid DNA of an individual colony was isolated with theQiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany)in accordance with the manufacturer's instructions and partly cleavedwith the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg,Germany, Product Description Sau3AI, Product No. 27-0913-02). The DNAfragments were dephosphorylated with shrimp alkaline phosphatase (RocheMolecular Biochemicals, Mannheim, Germany, Product Description SAP,Product No. 1758250). After separation by gel electrophoresis, thecosmid fragments in the size range of 1500 to 2000 bp were isolated withthe QiaExII Gel Extraction Kit (Product No. 20021, Qiagen, Hilden,Germany).

[0102] The DNA of the sequencing vector pZero-1, obtained from thecompany Invitrogen (Groningen, The Netherlands, Product Description ZeroBackground Cloning Kit, Product No. K2500-01) was cleaved with therestriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, ProductDescription BamHI, Product No. 27-0868-04). The ligation of the cosmidfragments in the sequencing vector pZero-1 was carried out as describedby Sambrook et al. (1989, Molecular Cloning: A laboratory Manual, ColdSpring Harbor) I.B.R., the DNA mixture being incubated overnight with T4ligase (Pharmacia Biotech, Freiburg, Germany).

[0103] This ligation mixture was then electroporated (Tauch et al. 1994,FEMS Microbiol Letters, 123:343-7) I.B.R. into the E. coli strainDH5αMCR (Grant, 1990, Proceedings of the National Academy of SciencesU.S.A., 87:4645-4649) I.B.R. and plated out on LB agar (Lennox, 1955,Virology, 1:190) with 50 μg/ml zeocin. The plasmid preparation of therecombinant clones was carried out with Biorobot 9600 (Product No.900200, Qiagen, Hilden, Germany).

[0104] The sequencing was carried out by the dideoxy chain-stoppingmethod of Sanger et al. (1977, Proceedings of the National Academy ofSciences U.S.A., 74:5463-5467) I.B.R. with modifications according toZimmermann et al. (1990, Nucleic Acids Research, 18:1067) I.B.R. The “RRdRhodamin Terminator Cycle Sequencing Kit” from PE Applied Biosystems(Product No. 403044, Weiterstadt, Germany) was used. The separation bygel electrophoresis and analysis of the sequencing reaction were carriedout in a “Rotiphoresis NF Acrylamide/Bisacrylamide” Gel (29:1) (ProductNo. A124.1, Roth, Karlsruhe, Germany) with the “ABI Prism 377” sequencerfrom PE Applied Biosystems (Weiterstadt, Germany).

[0105] The raw sequence data obtained were then processed using theStaden program package (1986, Nucleic Acids Research, 14:217-231)version 97-0 I.B.R. The individual sequences of the pZero1 derivativeswere assembled to a continuous contig. The computer-assisted codingregion analysis were prepared with the XNIP program (Staden, 1986,Nucleic Acids Research, 14:217-231) I.B.R. Further analyses were carriedout with the “BLAST search program” (Altschul et al., 1997, NucleicAcids Research, 25:3389-3402) I.B.R. against the non-redundant databankof the “National Center for Biotechnology Information” (NCBI, Bethesda,Md., USA) I.B.R.

[0106] The resulting nucleotide sequence of the zwa1 gene is shown inSEQ ID NO 1. Analysis of the nucleotide sequence showed an open readingframe of 597 base pairs, which was called the zwa1 gene. The zwa1 genecodes for a polypeptide of 199 amino acids, which is shown in SEQ ID NO2.

Example 3

[0107] Preparation of a Vector for Over-Expression of zwa1

[0108] From the strain ATCC 13032, chromosomal DNA was isolated by themethod of Eikmanns et al. (Microbiology 140: 1817-1828 (1994)) I.B.R. Onthe basis of the sequence of the zwa1 gene known for C. glutamicum fromexample 2, the following oligonucleotides were chosen for the polymerasechain reaction: zwa1-d1: 5′ TCA CA CCG ATG ATT CAG GC 3′ zwa1-d2: 5′ AGATTT AGC CGA CGA AAG CG 3′

[0109] The primers shown were synthesized by MWG Biotech (Ebersberg,Germany) and the PCR reaction was carried out by the standard PCR methodof Innis et al. (PCR protocols. A guide to methods and applications,1990, Academic Press) with Pwo-Polymerase from Boehringer. With the aidof the polymerase chain reaction, a DNA fragment approx. 1.0 kb in sizewas isolated, this carrying the zwa1 gene.

[0110] The amplified DNA fragment was ligated with the TOPO TA CloningKit from Invitrogen Corporation (Carlsbad, Calif., USA; Catalogue NumberK4500-01) in the vector pCR2.1-TOPO (Mead at al. (1991) Bio/Technology9:657-663) I.B.R. The E. coli strain Top10F′ was then electroporatedwith the ligation batch (Hanahan, In: DNA cloning. A practical approach.Vol. I. IRL-Press, Oxford, Washington D.C., USA) I.B.R. Selection forplasmid-carrying cells was made by plating out the transformation batchon LB agar (Sambrook et al., Molecular cloning: a laboratory manual.2^(nd) Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.) I.B.R., which had been supplemented with 25 mg/l kanamycin.

[0111] Plasmid DNA was isolated from a transformant with the aid of theQIAprep Spin Miniprep Kit from Qiagen and checked by restriction withthe restriction enzyme EcoRI and subsequent agarose gel electrophoresis(0.8%). The plasmid was called pCR2.1zwa1exp.

Example 4

[0112] Duplication of the zwa1 Gene in the Lysine Producer DSM 5715

[0113] The vector pCR2.1zwa1exp mentioned in example 6 waselectroporated by the electroporation method of Tauch et al. (FEMSMicrobiological Letters, 123:343-347 (1994)) I.B.R. in Corynebacteriumglutamicum DSM 5715. Strain DSM 5715 is anAEC(aminoethylcysteine)-resistant lysine producer. The vectorpCR2.1zwa1exp cannot replicate independently in DSM 5715 and is retainedin the cell only if it has integrated into the chromosome of DSM 5715.

[0114] Selection of clones with pCR2.1zwa1exp integrated into thechromosome was carried out by plating out the electroporation batch onLB agar (Sambrook et al., Molecular cloning: a laboratory manual. 2^(nd)Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989)I.B.R., which had been supplemented with 15 mg/l kanamycin. Fordetection of the integration, control PCR reactions were carried out bythe standard method of Innis et al. (PCR protocols. A guide to methodsand applications, 1990, Academic Press) I.B.R. with Pwo-Polymerase fromBoehringer. By combination of the primers zwa1-d1 and zwa1-d2 (cf.example 3) with the primers M13 universal forward(5′-gttttcccagtcacgac-3′) (Invitrogen Corporation, Cat. No. N540-02) andM13 universal reverse (5′-caggaaacagctatgac-3′) (Invitrogen Corporation,Cat. No. N530-02), which can bind only within the sequence of the vectorpCR2.1zwa1exp, it could be demonstrated that the plasmid pCR2.1zwa1exphad been inserted into the chromosome of the lysine producer DSM5715.The strain was called DSM5715::pCR2.1zwa1exp.

Example 5

[0115] Preparation of Lysine

[0116] The C. glutamicum strain DSM5715::pCR2.1zwa1exp obtained inexample 4 was cultured in a nutrient medium suitable for the productionof lysine and the lysine content in the culture supernatant wasdetermined.

[0117] For this, the strain was first incubated on an agar plate withthe corresponding antibiotic (brain-heart agar with kanamycin (25 mg/l)for 24 hours at 33° C. Starting from this agar plate culture, apreculture was seeded (10 ml medium in a 100 ml conical flask). Thecomplete medium CgIII was used as the medium for the preculture.Kanamycin (25 mg/l) was added to this. The preculture was incubated for48 hours at 33° C. at 240 rpm on a shaking machine. A main culture wasseeded from this preculture such that the initial OD (660 nm) of themain culture was 0.1. Medium MM was used for the main culture. Medium MMCSL (corn steep liquor) 5 g/l MOPS 20 g/l Glucose (autoclavedseparately) 50 g/l Salts: (NH₄)₂SO₄ 25 g/l KH₂PO₄ 0.1 g/l MgSO₄ * 7H₂O1.0 g/l CaCl₂ * 2H₂O 10 mg/l FeSO₄ * 7H₂O 10 mg/l MnSO₄ * H₂O 5.0 mg/lBiotin (sterile-filtered) 0.3 mg/l Thiamine * HCl (sterile-filtered) 0.2mg/l Leucine (sterile-filtered) 0.1 g/l CaCO₃ 25 g/l

[0118] The CSL, MOPS and the salt solution were brought to pH 7 withaqueous ammonia and autoclaved. The sterile substrate and vitaminsolutions were then added, as well as the CaCO₃ autoclaved in the drystate.

[0119] Culturing is carried out in a 10 ml volume in a 100 ml conicalflask with baffles. Kanamycin (25 mg/l) was added. Culturing was carriedout at 33° C. and 80% atmospheric humidity.

[0120] After 48 hours, the OD was determined at a measurement wavelengthof 660 nm with a Biomek 1000 (Beckmann Instruments GmbH, Munich). Ionexchange chromatography and post-column derivatization with ninhydrindetection determined the amount of lysine formed with an amino acidanalyzer from Eppendorf-BioTronik (Hamburg, Germany)

[0121] The result of the experiment is shown in table 1. TABLE 1 LysineHCl Strain OD (660) g/l DSM5715::pCR2.1zwa1exp 12.1 11.93 DSM5715 13.19.54

[0122] The abbreviations and designations used have the followingmeaning. ColE1 ori: Replication origin of the plasmid ColE1 lacZ: 5′ endof the β-galactosidase gene f1 ori: Replication origin of phage f1 KmR:Kanamycin resistance ApR: Ampicillin resistance EcoRI: Cleavage site ofthe restriction enzyme EcoRI zwa1 zwa1 gene

[0123] Further variations and modifications of the present inventionwill be apparent to those skilled in the art from a reading of theforegoing and are encompassed by the claims appended hereto.

[0124] German patent application 199 59 328.0 I.B.R. is relied upon andincorporated herein by reference.

1 4 1 1260 DNA Corynebacterium glutamicum -10_signal (383)..(388) 1ccgaaatatt ccaaatatgt aacataaatc acacccgatg attcaggcgg gatgacctgc 60gacttcaagg tcgcaccaaa gtcagattga tatagatttc gtaaataacg tgacacaatc 120gtgaccttcg ggttaccgtg tatcccaggc accgcaacag ttcatctgca agtccggctc 180atcgccaaac cctgtctggg gtcggaagtt gaacaacctc cttggtgcaa cagaacttta 240aaccacaaac tcccgcattc atgtgggcca tattgcagac agggacgggg aaaccaccca 300ccatcttttc acaaaagaag gcatggaggc caactccttg gggtgaagcc agacatccac 360tggcagagca actcctccgc tctaacccga cagctaacct cgacggcgac aa atg aga 418Met Arg 1 gga aaa ctt ttc atg gga cgt cac tcc act aag act agc tcc gcgttc 466 Gly Lys Leu Phe Met Gly Arg His Ser Thr Lys Thr Ser Ser Ala Phe5 10 15 acc aag ctc gca gct tcc acc atc gct ttc ggt gct gct gca acc atc514 Thr Lys Leu Ala Ala Ser Thr Ile Ala Phe Gly Ala Ala Ala Thr Ile 2025 30 atg gct cct tct gca tct gct gca cct gat tcc gac tgg gat cgc ctc562 Met Ala Pro Ser Ala Ser Ala Ala Pro Asp Ser Asp Trp Asp Arg Leu 3540 45 50 gca cag tgc gag tcc ggt ggt aac tgg gca atc aac acc ggt aac ggc610 Ala Gln Cys Glu Ser Gly Gly Asn Trp Ala Ile Asn Thr Gly Asn Gly 5560 65 tac cac ggt ggt ctg cag ttc tcc gct agc acc tgg gct gct tac ggc658 Tyr His Gly Gly Leu Gln Phe Ser Ala Ser Thr Trp Ala Ala Tyr Gly 7075 80 ggc cag gag ttc gct acc tac gca tac cag gca acc cgt gag cag cag706 Gly Gln Glu Phe Ala Thr Tyr Ala Tyr Gln Ala Thr Arg Glu Gln Gln 8590 95 atc gct gtt gca gag cgc acc ttg gct ggt cag ggc tgg ggc gca tgg754 Ile Ala Val Ala Glu Arg Thr Leu Ala Gly Gln Gly Trp Gly Ala Trp 100105 110 cct gct tgc tcc gct tcc ctt gga ctg aac tcc gct cca acc cag cgt802 Pro Ala Cys Ser Ala Ser Leu Gly Leu Asn Ser Ala Pro Thr Gln Arg 115120 125 130 gac ctc tcc gct acc acc tcc acc cca gag cca gct gca gct gcacca 850 Asp Leu Ser Ala Thr Thr Ser Thr Pro Glu Pro Ala Ala Ala Ala Pro135 140 145 gct gtt gct gag tac aac gct cct gca gcc aac atc gca gtt ggctcc 898 Ala Val Ala Glu Tyr Asn Ala Pro Ala Ala Asn Ile Ala Val Gly Ser150 155 160 acc gac ttg aac acc atc aag tcc acc tac ggc gct gtc acc ggcacc 946 Thr Asp Leu Asn Thr Ile Lys Ser Thr Tyr Gly Ala Val Thr Gly Thr165 170 175 ctc gct cag tac ggc atc acc gtt cca gct gag gtt gag tct tactac 994 Leu Ala Gln Tyr Gly Ile Thr Val Pro Ala Glu Val Glu Ser Tyr Tyr180 185 190 aac gct ttc gtc ggc taaatctagc tgcacttttt aaaagggagggaaccttaaa 1049 Asn Ala Phe Val Gly 195 cgggttccct ccctttttgc atgccatttcacgacgcgcc agtcatcctt ttgtgaattg 1109 ggcaccaaga tttcctgatt ttggccaccattttgccgaa accttggtgc cgaaagtacg 1169 cccagtagaa aaaccgcatg aaaaaagaggcaacaccgcc gaaacgggtt gcctcttttt 1229 taagtttctt agcggttgat ccgggtgtac g1260 2 199 PRT Corynebacterium glutamicum 2 Met Arg Gly Lys Leu Phe MetGly Arg His Ser Thr Lys Thr Ser Ser 1 5 10 15 Ala Phe Thr Lys Leu AlaAla Ser Thr Ile Ala Phe Gly Ala Ala Ala 20 25 30 Thr Ile Met Ala Pro SerAla Ser Ala Ala Pro Asp Ser Asp Trp Asp 35 40 45 Arg Leu Ala Gln Cys GluSer Gly Gly Asn Trp Ala Ile Asn Thr Gly 50 55 60 Asn Gly Tyr His Gly GlyLeu Gln Phe Ser Ala Ser Thr Trp Ala Ala 65 70 75 80 Tyr Gly Gly Gln GluPhe Ala Thr Tyr Ala Tyr Gln Ala Thr Arg Glu 85 90 95 Gln Gln Ile Ala ValAla Glu Arg Thr Leu Ala Gly Gln Gly Trp Gly 100 105 110 Ala Trp Pro AlaCys Ser Ala Ser Leu Gly Leu Asn Ser Ala Pro Thr 115 120 125 Gln Arg AspLeu Ser Ala Thr Thr Ser Thr Pro Glu Pro Ala Ala Ala 130 135 140 Ala ProAla Val Ala Glu Tyr Asn Ala Pro Ala Ala Asn Ile Ala Val 145 150 155 160Gly Ser Thr Asp Leu Asn Thr Ile Lys Ser Thr Tyr Gly Ala Val Thr 165 170175 Gly Thr Leu Ala Gln Tyr Gly Ile Thr Val Pro Ala Glu Val Glu Ser 180185 190 Tyr Tyr Asn Ala Phe Val Gly 195 3 19 DNA Corynebacteriumglutamicum 3 tcacaccgat gattcaggc 19 4 20 DNA Corynebacterium glutamicum4 agatttagcc gacgaaagcg 20

We claim:
 1. An isolated polynucleotide comprising a polynucleotidesequence chosen from the group consisting of: a) polynucleotide which isidentical to the extent of at least 70% to a polynucleotide which codesfor a polypeptide which comprises the amino acid sequence of SEQ ID NO2, b) polynucleotide which codes for a polypeptide which comprises anamino acid sequence which is identical to the extent of at least 70% tothe amino acid sequence of SEQ ID NO 2 c) polynucleotide which iscomplementary to the polynucleotides of a) or b), and d) polynucleotidecomprising at least 15 successive nucleotides of the polynucleotidesequences of a), b) or c).
 2. A polynucleotide according to claim 1,wherein the polynucleotide is a recombinant DNA which is capable ofreplication in coryneform bacteria.
 3. A polynucleotide according toclaim 1, wherein the polynucleotide is an RNA.
 4. A polynucleotideaccording to claim 2, comprising the nucleic acid sequence as shown inSEQ ID NO
 1. 5. A polynucleotide according to claim 2, which is DNAcapable of replication, comprising (i) the nucleotide sequence shown inSEQ ID NO 1, or (ii) at least one sequence which corresponds to asequence within the range of the degeneration of the genetic code of SEQID NO 1, or (iii) at least one sequence which hybridizes with a sequencecomplementary to sequences (i) or (ii), and (iv) sense mutations ofneutral function in (i).
 6. A vector comprising a polynucleotideaccording to claim 1, deposited in E. coli.
 7. The vector according toclaim 6, comprising the plasmid pCR2.1zwa1exp.
 8. Coryneform bacteriaserving as a host cell, comprising the vector according to claim 6, orin which the zwa1 gene is amplified.
 9. A process for the preparation ofL-amino acids comprising: a) fermenting an L-amino acid-producingbacteria in which at least the zwa1 gene is amplified.
 10. The processaccording to claim 9, and further comprising: b) concentrating theL-amino acid produced in the medium or in the cells of the bacteria. 11.The process according to claim 10, and further comprising: c) isolatingthe L-amino acid.
 12. The process according to claim 9, wherein theL-amino acid is L-lysine.
 13. The process according to claim 9, furthercomprising utilizing bacteria in which further genes of the biosynthesispathway of the L-amino acid are additionally amplified.
 14. The processaccording to claim 9, further comprising utilizing bacteria in which themetabolic pathways which reduce formation of L-lysine are at leastpartly eliminated.
 15. The process according to claims 9, wherein theL-amino acid-producing bacteria are coryneform bacteria.
 16. The processaccording to claim 9, wherein for the preparation of L-lysine, bacteriain which one or more genes chosen from the group consisting of 1) thedapA gene which codes for dihydrodipicolinate synthase, 2) the lysC genewhich codes for a feed back resistant aspartate kinase, 3) the dapD genewhich codes for tetradihydrodipicolinate succinylase, 4) the dapE genewhich codes for succinyl diaminopimelate desuccinylase, 5) the gap genewhich codes for glyceraldehyde 3-phosphate dehydrogenase, 6) the pycgene which codes for pyruvate carboxylase, 7) the mqo gene which codesfor malate:quinone oxidoreductase, and 8) the lysE gene which codes forlysine export, is/are amplified or overexpressed at the same time thebacteria are fermented.
 17. The process according to claim 9, whereinfor the preparation of L-lysine, bacteria in which one or more geneschosen from the group consisting of 1) the pck gene which codes forphosphoenol pyruvate carboxykinase or 2) the pgi gene which codes forglucose 6-phosphate isomerase is/are attenuated at the same time thebacteria are fermented.
 18. The process according to claim 9, whereinmicro-organisms of the genus Corynebacterium glutamicum are utilized.19. A method of using a polynucleotide sequence according to claim 1, ora part thereof, comprising hybridizing cDNA which codes for the Zwa1gene product with the polynucleotide or part thereof.
 20. A method ofusing a polynucleotide sequence according to claim 1, or a part thereof,comprising hybridizing cDNA having a higher than 70% similarity to apolynucleotide which codes for the Zwa1 gene.
 21. A method of using apolynucleotide sequence according to claim 1, or a part thereof,comprising hybridizing cDNA having a higher than 95% similarity to apolynucleotide which codes for the Zwa1 gene.